Biological Relevance:Enhancing blood supply is a common goal in the setting of ischemia due to injury or disease. The central pathway for cellular response to hypoxia is the hypoxia inducible factor (HIF)pathway. HIFs are transcription factors which activate genes encoding proteins that mediate adaptive responses to reduced oxygen availability (e.g. angiogenesis). The HIF complex consists of a heterodimer comprised of one of three alpha subunits (HIF-1alpha, HIF-2alpha, or HIF-3alpha)bound to the aryl hydrocarbon receptor nuclear translocator (ARNT), which is also known as HIF-1beta. Hypoxia induces changes in the accumulation and activity of the HIF-1alpha subunit. In most cells, the primary level of regulation is inhibition of the HIF-1alpha subunit degradation during hypoxia. Generally, the level of HIF-1alpha protein is low due to the on-going ubiquitination and proteosomal degradation. The molecular mechanism of degradation of HIF-1alpha subunits involves enzymatic prolyl hydroxylation on an oxygen degradation domain (ODD) which targets HIF-1alpha for ligation-mediated proteosomal degradation. The conserved proline residue is hydroxylated under normoxic conditions by one of a group of three prolyl hydroxylases (PHDs 1-3) . The reaction requires the presence of iron, oxygen and 2-oxoglutarate. Following prolyl hydroxylation, the HIF molecule is recognized by the Von Hippel Lindau (VHL) ubiquitinproteinligase. During hypoxia, prolyl hydroxylation is blocked, leading to HIF-1alphastabilization. A negative feedback loop is established whereby hypoxia and HIF-1alpha both upregulate PHD2 expression. This process results in accumulation of functional HIF-1alpha subunits, which dimerize with ARNT and translocate to the nucleus. In the nucleus, the HIF-1alpha/ARNT complex can couple with the coactivator p300, bind to hypoxia response elements (HREs) in the proximal promoter regions and transactivate HIF-sensitive genes which include metabolic pathways such as glut1 and angiogenic factors, such as VEGF. A further regulatory step exists, however. Hydroxylation of an asparagine residue by the factor inhibiting HIF (FIH) found within the nucleus can block the HIF/p300 association. The elucidation of these pathways has enabled strategies for experimental activation or inhibition of HIF.

Project Goal:Many disease processes and injuries have in common poor blood supply and/or lack of oxygen. The current proposal seeks to find agents that can activate the hypoxia inducible factor pathway which is responsible for initiating a response to low oxygen that includes metabolic adaptation and development of increased blood supply. These activators of the HIF pathway may be useful in a broad range of diseases such as heart disease and limb ischemia and in approaches to tissue regeneration. The current proposal seeks to find probes that can activate the hypoxia inducible factor pathway which is responsible for initiating a response to low oxygen that includes metabolic adaptation and development of increased blood supply.1)Probe attributes:a.Identify probes that activate HIF through mechanisms other then prolyl hydroxylation. b.Identify probes that more specifically target the HIF prolyl hydoroxylases (PHDs).c.Identify probes with higher potency than Desferrioxamine (EC50 of DFO is 17.8 muM).